Structural changes in human cytomegalovirus cytoplasmic assembly sites in the absence of UL97 kinase activity

Azzeh, Maysa; Honigman, Alik; Taraboulos, Albert; Rouvinski, Alexander; Wolf, Dana

doi:10.1016/j.virol.2006.05.037

Cited by 49 publications

(60 citation statements)

References 42 publications

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“…Marschall, unpublished data). Additional cytoplasmic functions of pUL97 (Kawaguchi et al, 1999;Prichard et al, 2005;Azzeh et al, 2006;Romaker et al, 2006) suggest a complex regulation system of pUL97 intracellular transport. Here, we investigated transiently expressed wild-type pUL97 in comparison to the two individually expressed large and small isoforms of pUL97.…”

Section: Results and Discussion Pul97 Occurs In Two Isoformsmentioning

confidence: 99%

Two isoforms of the protein kinase pUL97 of human cytomegalovirus are differentially regulated in their nuclear translocation

Webel

Milbradt

Auerochs

et al. 2010

Journal of General Virology

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The pUL97 protein kinase encoded by human cytomegalovirus is a multifunctional determinant of the efficiency of viral replication and phosphorylates viral as well as cellular substrate proteins. Here, we report that pUL97 is expressed in two isoforms with molecular masses of approximately 90 and 100 kDa. ORF UL97 comprises an unusual coding strategy in that five in-frame ATG start codons are contained within the N-terminal 157 aa. Site-directed mutagenesis, transient expression of point and deletion mutants and proteomic analyses accumulated evidence that the formation of the large and small isoforms result from alternative initiation of translation, with the start points being at amino acids 1 and 74, respectively. In vitro kinase assays demonstrated that catalytic activity, in terms of autophosphorylation and histone substrate phosphorylation, was indistinguishable for the two isoforms. An analysis of the intracellular distribution of pUL97 by confocal laser-scanning microscopy demonstrated that both isoforms have a pronounced nuclear localization. Surprisingly, mapping experiments performed to identify the nuclear localization signal (NLS) of pUL97 strongly suggest that the mechanism of nuclear transport is distinct for the two isoforms. While the extreme N terminus (large isoform) comprises a highly efficient, bipartite NLS (amino acids 6-35), a second sequence apparently conferring a less efficient mode of nuclear translocation was identified downstream of amino acid 74 (small and large isoforms). Taken together, the findings argue for a complex mechanism of nuclear translocation for pUL97 which might be linked with fine-regulatory differences between the two isoforms.

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Section: Results and Discussion Pul97 Occurs In Two Isoformsmentioning

confidence: 99%

Two isoforms of the protein kinase pUL97 of human cytomegalovirus are differentially regulated in their nuclear translocation

Webel

Milbradt

Auerochs

et al. 2010

Journal of General Virology

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“…Interestingly, the function of UL97 in promoting replication during infection with clinical strains largely depends on the presence of UL135. Further, UL97 mutant viruses exhibit defects in the late stages of virus maturation, producing a number of empty virions and defects in secondary envelopment (55)(56)(57)(58). The similarities in phenotypes associated with the loss of UL135 or UL97 and the interplay between UL135 and UL97 during infection that these phenotypes suggest are intriguing.…”

Section: Discussionmentioning

confidence: 99%

Antagonistic Determinants Controlling Replicative and Latent States of Human Cytomegalovirus Infection

Umashankar

Rak

Bughio

et al. 2014

J Virol

139

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The mechanisms by which viruses persist and particularly those by which viruses actively contribute to their own latency have been elusive. Here we report the existence of opposing functions encoded by genes within a polycistronic locus of the human cytomegalovirus (HCMV) genome that regulate cell type-dependent viral fates: replication and latency. The locus, referred to as the UL133-UL138 (UL133/8) locus, encodes four proteins, pUL133, pUL135, pUL136, and pUL138. As part of the ULb= region of the genome, the UL133/8 locus is lost upon serial passage of clinical strains of HCMV in cultured fibroblasts and is therefore considered dispensable for replication in this context. Strikingly, we could not reconstitute infection in permissive fibroblasts from bacterial artificial chromosome clones of the HCMV genome where UL135 alone was disrupted. The loss of UL135 resulted in complex phenotypes and could ultimately be overcome by infection at high multiplicities. The requirement for UL135 but not the entire locus led us to hypothesize that another gene in this locus suppressed virus replication in the absence of UL135. The defect associated with the loss of UL135 was largely rescued by the additional disruption of the UL138 latency determinant, indicating a requirement for UL135 for virus replication when UL138 is expressed. In the CD34 ؉ hematopoietic progenitor model of latency, viruses lacking only UL135 were defective for viral genome amplification and reactivation. Taken together, these data indicate that UL135 and UL138 comprise a molecular switch whereby UL135 is required to overcome UL138-mediated suppression of virus replication to balance states of latency and reactivation. IMPORTANCEMechanisms by which viruses persist in their host remain one of the most poorly understood phenomena in virology. Herpesviruses, including HCMV, persist in an incurable, latent state that has profound implications for immunocompromised individuals, including transplant patients. Further, the latent coexistence of HCMV may increase the risk of age-related pathologies, including vascular disease. The key to controlling or eradicating HCMV lies in understanding the molecular basis for latency. In this work, we describe the complex interplay between two viral proteins, pUL135 and pUL138, which antagonize one another in infection to promote viral replication or latency, respectively. We previously described the role of pUL138 in suppressing virus replication for latency. Here we demonstrate a role of pUL135 in overcoming pUL138-mediated suppression for viral reactivation. From this work, we propose that pUL135 and pUL138 constitute a molecular switch balancing states of latency and reactivation.

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“…This is in contrast to RV-VM1, where pp65 was exclusively located in the nucleus and no cytoplasmic aggregates were found. Azzeh et al (2006) showed that infection of HFF with a pUL97-deletion mutant or inhibition of pUL97 in wt-HCMVinfected cells by NGIC-I (non-glyosidic indolocarbazole I) resulted in a reorganization of cytoplasmic assembly sites, including a redistribution of the late protein pp28 and the formation of 2-8 mm wide vacuoles. We observed neither the formation of vacuoles in the cytoplasm nor the redistribution of pp28 after infection with RV-VM1 (not shown).…”

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confidence: 99%

Modification of the major tegument protein pp65 of human cytomegalovirus inhibits virus growth and leads to the enhancement of a protein complex with pUL69 and pUL97 in infected cells

Becke

Fabre-Mersseman

Aue

et al. 2010

Journal of General Virology

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The tegument protein pp65 of human cytomegalovirus (HCMV) is abundant in lytically infected human foreskin fibroblasts (HFF), as well as in virions and subviral dense bodies (DB). Despite this, we showed previously that pp65 is dispensable for growth in HFF. In the process of refining a DB-based vaccine candidate, different HCMV mutants were generated, expressing a dominant HLA-A2-presented peptide of the IE1 protein fused to pp65. One of the mutant viruses (RV-VM1) surprisingly showed marked impairment in virus release from HFF. We hypothesized that analysis of the phenotypic alterations of RV-VM1 would provide insight into the functions of pp65, poorly defined thus far. RV-VM1 infection resulted in nuclear retention of the fusion protein and reorganization of nuclear inclusion bodies. Coimmunoprecipitation experiments suggested that wild-type (wt) pp65 and pp65-VM1 were substrates of the viral pUL97 kinase in vitro and formed a complex with the viral RNA-export protein pUL69 and with pUL97 in lysates of infected cells. No evidence for an impairment of pUL97 within this complex was found. However, RV-VM1 replication in infected cells was resistant to a pUL97 inhibitor, and pUL97 inhibitors mimicked the mutant in terms of pp65 being retained in the nucleus. The results suggest that the life cycle of RV-VM1 was impeded at the stages of early-late transcription, RNA export or capsid maturation. wt-pp65 may play a role at these stages of infection, and complex formation with pUL69 and pUL97 may be important for that function. INTRODUCTIONHuman cytomegalovirus (HCMV) is a ubiquitous pathogen that severely affects individuals with impaired or immature immune-defence functions (Griffiths et al., 2009;Mocarski et al., 2007). The components of the HCMV tegument have recently attracted considerable attention, as these proteins carry functions important for various stages of the virus replication cycle (reviewed by Kalejta, 2008). The phosphoprotein pp65 (ppUL83) has long been known as an abundant tegument constituent (Roby & Gibson, 1986), yet it is dispensable for HCMV growth in human foreskin fibroblast (HFF) cell cultures (Schmolke et al., 1995b).pp65 is an important target antigen of cellular and humoral immune responses (Beninga et al., 1995; McLaughlinTaylor et al., 1994; Plachter et al., 1990;Wills et al., 1996). It would thus be reasonable to assume that modification or deletion of the protein would be favourable for the virus. However, the sequence of pp65 is highly conserved in laboratory strains and clinical isolates (Dolan et al., 2004;Pande et al., 1991) and lack of pp65 expression in such strains has never been reported. Indeed, detection of pp65 has been used as a reliable diagnostic marker for acute HCMV infection in transplant recipients for over 15 years (Grefte et al., 1992a, b Initial studies described an association of pp65 with serine/ threonine kinase activity (Britt & Auger, 1986;Somogyi et al., 1990). Subsequent analyses identified the cellular Polo-like kinase 1 (Gallina et al., 1999) and the vi...

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Structural changes in human cytomegalovirus cytoplasmic assembly sites in the absence of UL97 kinase activity

Cited by 49 publications

References 42 publications

Two isoforms of the protein kinase pUL97 of human cytomegalovirus are differentially regulated in their nuclear translocation

Two isoforms of the protein kinase pUL97 of human cytomegalovirus are differentially regulated in their nuclear translocation

Antagonistic Determinants Controlling Replicative and Latent States of Human Cytomegalovirus Infection

Modification of the major tegument protein pp65 of human cytomegalovirus inhibits virus growth and leads to the enhancement of a protein complex with pUL69 and pUL97 in infected cells

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